PROJECT SUMMARY Ticks require a blood meal before molting at each developmental stage and for adult females to produce eggs. This obligatory reliance on vertebrate blood makes them ideal vectors for pathogens of a wide variety of diseases. Lyme disease (LD), caused by the bacteria Borrelia burgdorferi (Bb) and transmitted by the black- legged tick, Ixodes scapularis, is the most significant vector-borne disease in the United States. Over 300,000 LD cases are diagnosed in the US annually. Increased incidence and distribution of LD and other tick-borne diseases necessitates a better understanding of vector biology to develop new approaches for vector control. Recent advances in genetic transformation technology such as clustered, regularly interspaced, short, palindromic repeats (CRISPR-Cas9), now permit the relationships between specific genes and their function to be elucidated in non-model arthropods. These tools are ideal for dissecting tick molecular biology and vector- pathogen interactions. However, to date, several technical hurtles have prevented these techniques from being applied to study tick molecular biology. We propose to develop the first CRISPR-Cas9 tools to knockout genes in ticks. As a proof of concept, we will target tick insulin-like peptides (ILPs) and the insulin receptor. This fits well with our long-term research goal of understanding neuroendocrine regulation of tick reproduction and host- seeking. We hypothesize that tick ILPs play key roles in nutrient metabolism and reproduction in a manner similar to ILPs in mosquitoes. Since the original submission of this proposal, we have developed techniques for successful tick embryo injection; however, fungal contamination of embryos resulted in low survival. We now aim to optimize embryo injection and rearing protocols (Aim 1). In order to identify promoters for use in I. scapularis transformation and gene expression, we will be evaluating promoters in a tick cell line (Aim 2). Our newly developed embryo injection protocol will pave the road for CRISPR-Cas9 based editing of insulin signaling pathway genes (Aim 3). The expected outcomes of our work will provide new tools to determine the genetic basis of many tick phenotypes, including those involved with transmission of disease. Ticks transmit the widest variety of pathogens of any blood feeding arthropod, including bacteria, rickettsiae, protozoa, and viruses. Our work will impact the tick research community by facilitating genetic transformations that are required to overcome a current barrier in the field.